In some retinal disorders including atrophic forms of macular degeneration, the death of retinal pigment epithelial (RPE) cells is an early and crucial event leading to visual impairment. Work in the laboratory is aimed at determining whether the RPE cells at risk are those that have accumulated critical levels of lipofuscin pigment. Indeed, it has been shown that a major constituent of RPE lipofuscin, the fluorophore A2E, can mediate blue light damage and can exert a detergent-like activity on cell membranes. To track the events that lead to the death of blue light-irradiated A2E-laden cultured RPE, the involvement of singlet oxygen and the formation of reactive photoxidative products of A2E are being investigated. DNA is being explored as a target of these reactive intermediates, and the ability of antioxidants such as vitamin E to protect against blue light damage to A2E-laden RPE is being tested. Having shown that the cell death program involves activation of caspase proteinases and modulation by the mitochondria protein Bcl-2, work is now directed towards identifying caspase-9 as the initiator caspase. In terms of the upstream signaling that leads to activation of the cell death program, activation of p53 in association with DNA damage is being investigated, as are components of the stress kinase pathway. The role of pro-apoptotic proteins, which associate with mitochondria, are also being studied. A2E accumulates in the lysosomal compartment of the cell and even in the absence of illumination, can damage RPE cells when amassed to certain levels. This effect is being investigated in terms of a detergent-like disruption of the lysosomal membrane. Structural features of A2E that determine this behavior are also to be examined. In studies of A2E biosynthesis, conditions (bright light, antioxidant-treatment, NADPH depletion) under which the formation of A2E may be accelerated or impaired are being probed. The premise that A2E is generated from the precursor A2-PE by enzyme-mediated mechanisms within the lysosomal compartment of the RPE cell is also being examined. To revisit questions related to the composition of human RPE lipofuscin, additional photoisomers of A2E are being identified and the presence of all-trans-retinal condensation products other than A2E is being explored. The long-term goals of this work are to develop therapies that would reduce the formation of A2E, destroy the formed molecule within the RPE cell, or counter the deleterious effects of its accumulation.